The function of the mature nervous system is critically dependent on the intricate neuronal network. The ultimate objective of this study is to understand mechanisms by which axonal projections are determined during development. Recent studies have revealed that a few protein families participate in determining when and where these neuronal connections form. However, we are just at the beginning of understanding the underlying mechanisms of these proteins functioning in axon pathfinding. Semaphorins have been demonstrated to be capable of steering developing axons. Our previous studies have led to the identification of two families of transmembrane proteins, namely neuropilins and plexins, as required cell surface receptors for members of semaphorins, and provided a unique entry point to explore the signaling mechanism leading to the cytoskeletal rearrangements underlying the axon guidance effects of semaphorins. In this study we propose to use neuropilins and plexins as probes to explore the signaling mechanisms which mediate the axon guidance effect of Semaphorin3A (Sema3A), a prototype of vertebrate semaphorins. Specifically, we will address the following questions: 1). How the functional specificity of semaphorins is determined at the receptor level? We will address this question by determining which plexins act as co-receptors of neuropilin-1 to mediate the Sema3A activity; 2). What is the molecular mediators between the neuropilin/plexin receptor complex and the intracellular cytoskeletal machinery? Recent studies indicated that plexins could be phosphorylated at tyrosine residues, suggesting a potentially important event in the signaling pathway. Thus we will investigate whether the tyrosine phosphorylation of plexins is critical in relaying Sema3A signal from the receptor to the interior of cells; 3). What is the role of neuropilin-1 in the Sema3A signaling pathway? In addition to its well-defined ligand binding activity, we have recently identified a PDZ domain-containing protein GIPC that can bind to the intracellular domain of neuropilin-l and is required for neuronal responses to Sema3A. In light of the fact that other PDZ proteins act in determining the subcellular localization of their interacting proteins, we will determine whether the GIPC-mediated connection affects the localization of neuropilin-1 and thus regulates the axonal responses to Sema3A. These studies together should deepen and broaden our understanding of how semaphorins are involved in axon guidance during development, and may lend insights into the inability of adult central nervous system to regenerate, and help devise treatments for brain and spinal cord injuries.